Concomitant assessment of PD-1 and CD56 expression identifies subsets of resting cord blood Vδ2 T cells with disparate cytotoxic potential.

Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.

Prevalence and risk factors of detectable HIV viral load among pregnant women with HIV infection seeking antenatal care in Southern Malawi.

We evaluated detectable viral load (VL) in pregnant women established on antiretroviral therapy (ART) for at least 6 months before conception and those self-reported as ART naïve at first antenatal care (ANC) at two government clinics in Southern Malawi. We used logistic regression to identify the predictors of detectable viral load (VL), defined as any measure greater than 400 copies/ml. Of 816 women, 67.9% were established on ART and 32.1% self-reported as ART naïve. Among women established on ART, 10.8% had detectable VL and 9.9% had VL >1000 copies/ml (WHO criteria for virological failure). In adjusted analysis, among women established on ART, virological failure was associated with younger age (p = .02), "being single/widowed" (p = 0.001) and no previous deliveries (p = .05). One fifth of women who reported to be ART-naive were found to have an undetectable VL at first ANC. None of the demographic factors could significantly differentiate those with high versus low VL in the ART-naïve sub-sample. In this cohort, approximately 90% of women who had initiated ART prior to conception had an undetectable VL at first ANC. This demonstrates good success of the ART program but identifies high risk populations that require additional support.

Effective Barriers: The Role of NKT Cells and Innate Lymphoid Cells in the Gut.

The critical role of commensal microbiota in regulating the host immune response has been established. In addition, it is known that host-microbial interactions are bidirectional, and this interplay is tightly regulated to prevent chronic inflammatory disease. Although many studies have focused on the role of classic T cell subsets, unconventional lymphocytes such as NKT cells and innate lymphoid cells also contribute to the regulation of homeostasis at mucosal surfaces and influence the composition of the intestinal microbiota. In this review, we discuss the mechanisms involved in the cross-regulation between NKT cells, innate lymphoid cells, and the gut microbiota. Moreover, we highlight how disruptions in homeostasis can lead to immune-mediated disorders.

Prevalence and characteristics of metabolic syndrome and its components among adults living with and without HIV in Nigeria: a single-center study.

BACKGROUND: Persons living with HIV (PLHIV) now live longer due to effective combination antiretroviral therapy. However, emerging evidence indicates that they may be at increased risk for some cardiometabolic disorders. We compared the prevalence of metabolic syndrome (MetS) and its component disorders between persons living with and without HIV in Nigeria. METHODS: This was a cross-sectional analysis of baseline data from a prospective cohort study of non-communicable diseases among PLHIV along with age- and sex-matched persons without HIV (PWoH) at the University of Abuja Teaching Hospital Nigeria. We collected sociodemographic and clinical data, including anthropometric measures and results of relevant laboratory tests. MetS was defined using a modification of the third report of the National Cholesterol Education Program Adult Treatment Panel (NCEP ATP III) criteria. RESULTS: Of the 440 PLHIV and 232 PWoH, women constituted 50.5% and 51.3% respectively. The median age of the PLHIV was 45 years while that of the PWoH was 40 years. The prevalence of MetS was 30.7% (95% CI: 26.4%, 35.2%) and 22.8% (95% CI: 17.6%, 28.8%) among the PLHIV and PWoH respectively (P = 0.026). Independent associations were found for older age (P < 0.001), female sex (P < 0.001), family history of diabetes (P < 0.001), family history of hypertension (P = 0.013) and alcohol use (P = 0.015). The prevalence of component disorders for PLHIV versus PWoH were as follows: high blood pressure (22.3% vs 20.3%), prediabetes (33.8% vs 21.1%), diabetes (20.5% vs 8.2%), high triglycerides (24.5% vs 17.2%), low HDL-Cholesterol (51.1% vs 41.4%), and abdominal obesity (38.4% vs 37.1%). Adjusting for age and sex, prediabetes, diabetes, and low HDL-Cholesterol were significantly associated with HIV status. Duration on antiretroviral therapy, protease inhibitor-based regimen, CD4 count, and viral load were associated with some of the disorders mostly in unadjusted analyses. CONCLUSION: We found a high burden of MetS and its component disorders, with significantly higher prevalence of dysglycemia and dyslipidemia among PLHIV as compared to PWoH. Integration of strategies for the prevention and management of MetS disorders is needed in HIV treatment settings.

Age-related changes in PD-1 expression coincide with increased cytotoxic potential in Vδ2 T cells during infancy.

Human Vγ9Vδ2 T cells respond to several diverse pathogens by sensing microbial cholesterol intermediates. Unlike CD4 T cells, they are poised for rapid Th1-like responses even before birth, which allows them to play a key role in the first line of defense against pathogens in early life. However, their regulation and functional maturation during infancy (in particular the acquisition of cytotoxic potential) remain understudied. We thus characterized their responses to cholesterol intermediates and Bacille Calmette-Guérin in a cohort of African neonates and 12-month-old infants. Infant Vδ2 lymphocytes exhibited intermediate or adult-like expression of markers associated with differentiation or function, intermediate proliferative responses, and adult-like cytotoxic potential. The enhancement of Vδ2 cell cytotoxic potential coincided with decreasing PD-1 and increasing NKG2A expression. Our results are consistent with the hypothesis that switching from a PD-1+ to a NKG2A+ phenotype during infancy indicates a shift in mechanisms regulating Vδ2 T cell function.

Persistent gamma delta T-cell dysfunction in chronic HCV infection despite direct-acting antiviral therapy induced cure.

Immune dysfunction is a hallmark of chronic HCV infection and viral clearance with direct antivirals recover some of these immune defects. TCRVγ9Vδ2 T-cell dysfunction in treated HCV patients however is not well studied and was the subject of this investigation. Peripheral blood cells from patients who had achieved sustained virologic response (SVR) or those who had relapsed after interferon-free therapy were phenotyped using flow cytometry. Functional potential of Vγ9Vδ2 T cells was tested by measuring proliferation in response to aminobisphosphonate zoledronic acid, and cytotoxicity against HepG2 hepatoma cell line. TCR sequencing was performed to analyse impact of HCV infection on Vδ2 T-cell repertoire. Vγ9Vδ2 cells from patients were activated and therapy resulted in reduction of CD38 expression on these cells in SVR group. Relapsed patients had Vδ2 cells with persistently activated and terminally differentiated cytotoxic phenotype (CD38+ CD45RA+ CD27- CD107a+ ). Irrespective of outcome with therapy, majority of patients had persistently poor Vδ2 T-cell proliferative response to zoledronate along with lower expression of CD56, which identifies anti-tumour cytotoxic subset, relative to healthy controls. There was no association between the number of antigen reactive Vγ2-Jγ1.2 TCR rearrangements at baseline and levels of proliferation indicating nonresponse to zoledronate is not due to depletion of phosphoantigen responding chains. Thus, HCV infection results in circulating Vγ9Vδ2 T cells with a phenotype equipped for immediate effector function but poor cytokine response and expansion in response to antigen, a functional defect that may have implications for susceptibility for carcinogenesis despite HCV cure.

Prolonged PD1 Expression on Neonatal Vδ2 Lymphocytes Dampens Proinflammatory Responses: Role of Epigenetic Regulation.

A successful pregnancy depends on the maintenance of tolerance at the fetal-maternal interface; strong inflammation in the placental bed is generally associated with adverse fetal outcomes. Among the mechanisms that foster tolerance and limit inflammation, the fetal immune system favors Th2 or regulatory responses over Th1 responses. The unintended consequence of this functional program is high susceptibility to infections. Human Vδ2 T cells mount innate-like responses to a broad range of microorganisms and are poised for Th1 responses before birth. In infants they likely play a key role in protection against pathogens by exerting early Th1 effector functions, improving function of other innate cells, and promoting Th1 polarization of adaptive responses. However, their propensity to release Th1 mediators may require careful regulation during fetal life to avoid exaggerated proinflammatory responses. We investigated molecules with the potential to act as a rheostat for fetal Vδ2 cells. Programmed death 1 (PD1) is a negative regulator of T cell responses and a determinant of tolerance, particularly at the fetal-maternal interface. Neonatal Vδ2 cells upregulate PD1 shortly after activation and, unlike their adult counterparts, express this molecule for at least 28 d. Engagement of PD1 by one of its ligands, PDL1, effectively dampens TCR-mediated responses (TNF-α production and degranulation) by neonatal Vδ2 cells and may thus help maintain their activity within safe limits. PD1 expression by neonatal Vδ2 cells is inversely associated with promoter DNA methylation. Prolonged PD1 expression may be part of a functional program to control Vδ2 cell inflammatory responses during fetal life.

Evolution and function of the TCR Vgamma9 chain repertoire: It's good to be public.

Lymphocytes expressing a T cell receptor (TCR) composed of Vgamma9 and Vdelta2 chains represent a minor fraction of human thymocytes. Extrathymic selection throughout post-natal life causes the proportion of cells with a Vgamma9-JP rearrangement to increase and elevates the capacity for responding to non-peptidic phosphoantigens. Extrathymic selection is so powerful that phosphoantigen-reactive cells comprise about 1 in 40 circulating memory T cells in healthy adults and the subset expands rapidly upon infection or in response to malignancy. Skewing of the gamma delta TCR repertoire is accompanied by selection for public gamma chain sequences such that many unrelated individuals overlap extensive in their circulating repertoire. This type of selection implies the presence of a monomorphic antigen-presenting molecule that is an object of current research but remains incompletely defined. While selection on a monomorphic presenting molecule may seem unusual, similar mechanisms shape the alpha beta T cell repertoire including the extreme examples of NKT or mucosal-associated invariant T cells (MAIT) and the less dramatic amplification of public Vbeta chain rearrangements driven by individual MHC molecules and associated with resistance to viral pathogens. Selecting and amplifying public T cell receptors whether alpha beta or gamma delta, are important steps in developing an anticipatory TCR repertoire. Cell clones expressing public TCR can accelerate the kinetics of response to pathogens and impact host survival.

Cord blood Vγ2Vδ2 T cells provide a molecular marker for the influence of pregnancy-associated malaria on neonatal immunity.

BACKGROUND: Plasmodium falciparum placental infection primes the fetal immune system and alters infant immunity. Mechanisms leading to these outcomes are not completely understood. We focused on Vγ2Vδ2 cells, which are part of the immune response against many pathogens, including P. falciparum. These unconventional lymphocytes respond directly to small, nonpeptidic antigens, independent of major histocompatibility complex presentation. We wondered whether placental malaria, which may increase fetal exposure to P. falciparum metabolites, triggers a response by neonatal Vγ2Vδ2 lymphocytes that can be a marker for the extent of fetal exposure to malarial antigens. METHODS: Cord blood mononuclear cells were collected from 15 neonates born to mothers with P. falciparum infection during pregnancy (8 with placental malaria) and 25 unexposed neonates. Vγ2Vδ2 cell phenotype, repertoire, and proliferative responses were compared between newborns exposed and those unexposed to P. falciparum. RESULTS: Placental malaria-exposed neonates had increased proportions of central memory Vγ2Vδ2 cells in cord blood, with an altered Vγ2 chain repertoire ex vivo and after stimulation. CONCLUSION: Our results suggest that placental malaria affects the phenotype and repertoire of neonatal Vγ2Vδ2 lymphocytes. Placental malaria may lower the capacity for subsequent Vγ2Vδ2 cell responses and impair the natural resistance to infectious diseases or the response to pediatric vaccination.

Vγ2Vδ2 T cell Costimulation Increases NK cell Killing of Monocyte-derived Dendritic Cells.

Interactions between NK and dendritic cells (DC) affect maturation and function of both cell populations, including NK killing of DC (editing) that is important for controlling the quality of immune responses. We also know that antigen-stimulated Vγ2Vδ2 T cells costimulate NK cells via 4-1BB to enhance killing of tumor cell lines but we do not know what regulates 4-1BB expression or whether other NK effector functions including DC killing, might also be influenced by NK:γδ T cell cross talk. Here we show that antigen-stimulated γδ T cells costimulate NK through ICOS:ICOSL and this signal increases NK killing of autologous DC. Effects of NK:γδ T cell co-culture, which could be reproduced with soluble ICOS-Fc fusion protein, included increased CD69 and 4-1BB expression, IFN-γ, TNF-α, MIP-1ß, I-309, RANTES and sFasL production, as well as elevated mRNA levels for costimulatory receptors OX40 (TNFRSF4) and GITR (TNFRSF18). Thus, ICOS/ICOSL costimulation of NK by Vγ2Vδ2 T cells had broad effects on NK phenotype and effector functions. The NK γδ T cell cross talk links innate and antigen-specific lymphocyte responses in the control of cytotoxic effector function and dendritic cell killing. This article is protected by copyright. All rights reserved.

Human cord blood γδ T cells expressing public Vγ2 chains dominate the response to bisphosphonate plus interleukin-15.

Compared with adults, the circulating Vγ2Vδ2 T-cell population in cord blood is present at low levels and does not show the strong bias for Vγ2-Jγ1.2 rearrangements. These features may be a result of limited exposure to stimulatory phosphoantigens, lack of T-cell-derived interleukin-2 (IL-2) or both. In cord blood mononuclear cell cultures, a single round of stimulation, using aminobisphosphonates to elevate phosphoantigen levels, resulted in expansion of adult-like Vγ2 chains and accumulation of memory cells with cytotoxic potential. Selection was similar using IL-2 or myeloid-derived IL-15. The Vγ2Vδ2 T cells present in neonates are capable of generating potent immune responses even when relying on IL-15.

RTS,S today and tomorrow's science.

The first malaria vaccine has been recently approved for children living in malaria-endemic areas. While this is long-awaited and welcome news, the modest efficacy of the vaccine highlights several areas that require further attention. Here, we describe the likely impact of the vaccine and where clinical and basic discovery research will still be required.

The Association among Malaria in Pregnancy, Neonatal inflammation, and Neurocognitive Development in a Cohort of Malawian Infants.

Malaria in pregnancy (MIP) causes poor birth outcomes, but its impact on neurocognitive development has not been well characterized. Between 2012 and 2014, we enrolled 307 mother-infant pairs and monitored 286 infants for neurocognitive development using the Malawi Developmental Assessment Tool at 6, 12, and 24 months of age. MIP was diagnosed from peripheral blood and placental specimens. Cord blood cytokine levels were assessed for a subset of neonates. Predictors of neurodevelopment were examined using mixed-effect logistic regression for developmental delay. Among the participants, 78 mothers (25.4%) had MIP, and 45 infants (15.7%) experienced severe neurocognitive delay. MIP was not associated with differences in cord blood cytokine levels or neurocognitive development. Preterm birth, low birthweight, increasing maternal education level, and increasing interleukin 6 levels were associated significantly with delay. The results highlight the prevalence of severe delay and a need for broad access to early childhood support in this setting.

HIV-Associated Interactions Between Oral Microbiota and Mucosal Immune Cells: Knowledge Gaps and Future Directions.

Even with sustained use of antiretroviral therapy (ART), HIV-infected individuals have an increased risk of systemic comorbid conditions and oral pathologies, including opportunistic infections, oral mucosal inflammation, and gingival and periodontal diseases. The immune-mediated mechanisms that drive this increased risk, in the context of sustained viral suppression, are unclear. HIV infection, even when controlled, alters microbial communities contributing to a chronic low-grade inflammatory state that underlies these non-HIV co-morbidities. The higher prevalence of dental caries, and mucosal and periodontal inflammation reported in HIV-infected individuals on ART is often associated with differentially abundant oral microbial communities, possibly leading to a heightened susceptibility to inflammation. This mini-review highlights current gaps in knowledge regarding the microbe-mediated oral mucosal immunity with HIV infection while discussing opportunities for future research investigations and implementation of novel approaches to elucidate these gaps. Interventions targeting both inflammation and microbial diversity are needed to mitigate oral inflammation-related comorbidities, particularly in HIV-infected individuals. More broadly, additional research is needed to bolster general models of microbiome-mediated chronic immune activation and aid the development of precise microbiota-targeted interventions to reverse or mitigate adverse outcomes.

Maternal HIV status skews transcriptomic response in infant cord blood monocytes exposed to Bacillus Calmette--Guerín.

OBJECTIVES: HIV-exposed uninfected (HEU) infants exhibit altered vaccine responses and an increased mortality compared with HIV-unexposed infants. Here, vaccine responses in HEU and HIV-unexposed cord blood monocytes (CBMs) were assessed following Bacillus Calmette--Guerín (BCG) treatment. DESIGN: Innate responses to in-vitro BCG treatment were assessed through transcriptional profiling using CBMs obtained from a Nigerian cohort of HIV-infected and uninfected women. METHODS: HIV-unexposed (n = 9) and HEU (n = 10) infant CBMs were treated with BCG and transcriptionally profiled with the Nanostring nCounter platform. Differential expression and pathway enrichment analyses were performed, and transcripts were identified with enhanced or dampened BCG responses. RESULTS: Following BCG stimulation, several pathways associated with inflammatory gene expression were upregulated irrespective of HIV exposure status. Both HIV-unexposed and HEU monocytes increased expression of several cytokines characteristic of innate BCG responses, including IL1ß, TNFα, and IL-6. Using differential expression analysis, we identified genes significantly upregulated in HEU compared with HIV-unexposed monocytes including monocyte chemokine CCL7 and anti-inflammatory cytokine TNFAIP6. In contrast, genes significantly upregulated in HIV-unexposed compared with HEU monocytes include chemokine CCL3 and cytokine IL23A, both of which influence anti-mycobacterial T-cell responses. Finally, two genes, which regulate prostaglandin production, CSF2 and PTGS2, were also more significantly upregulated in the HIV-unexposed cord blood indicating that inflammatory mediators are suppressed in the HEU infants. CONCLUSION: HEU monocytes exhibit altered induction of several key innate immune responses, providing mechanistic insights into dysregulated innate response pathways that can be therapeutically targeted to improve vaccine responses in HEU infants.

Control of CD56 expression and tumor cell cytotoxicity in human Vgamma2Vdelta2 T cells.

BACKGROUND: In lymphocyte subsets, expression of CD56 (neural cell adhesion molecule-1) correlates with cytotoxic effector activity. For cells bearing the Vgamma2Vdelta2 T cell receptor, isoprenoid pyrophosphate stimulation leads to uniform activation and proliferation, but only a fraction of cells express CD56 and display potent cytotoxic activity against tumor cells. Our goal was to show whether CD56 expression was regulated stochastically, similar to conventional activation antigens, or whether CD56 defined a lineage of cells committed to the cytotoxic phenotype. RESULTS: Tracking individual cell clones defined by their Vgamma2 chain CDR3 region sequences, we found that CD56 was expressed on precursor cytotoxic T cells already present in the population irrespective of their capacity to proliferate after antigen stimulation. Public T cell receptor sequences found in the CD56+ subset from one individual might appear in the CD56- subset of another donor. The commitment of individual clones to CD56+ or CD56- lineages was stable for each donor over a 1 year interval. CONCLUSION: The ability to express CD56 was not predicted by TCR sequence or by the strength of signal received by the TCR. For gammadelta T cells, cytotoxic effector function is acquired when cytotoxic precursors within the population are stimulated to proliferate and express CD56. Expression of CD56 defines a committed lineage to the cytotoxic phenotype.

Impacts of HIV infection on Vgamma2Vdelta2 T cell phenotype and function: a mechanism for reduced tumor immunity in AIDS.

HIV infection causes rapid and lasting defects in the population of Vgamma2Vdelta2 T cells. To fully describe the impact of HIV, we examined PBMC samples from HIV+ patients receiving highly active antiretroviral therapy, who had displayed prolonged viral control and CD4 counts above 300 cells/mm3. We observed lower frequencies of CD27-/CD45RA- Vgamma2Vdelta2 cells in HIV+ individuals when compared with controls, coupled with an increased proportion of CD45RA+ cells. These changes were common among 24 HIV+ patients and were not related to CD4 cell count or viral RNA burden. Vgamma2 cells from HIV+ individuals had lower expression of Granzyme B and displayed reduced cytotoxicity against Daudi targets after in vitro stimulation. There was increased expression of FasR (CD95) on Vgamma2 cells from HIV+ PBMC that may be a mechanism for depletion of Vgamma2 cells during disease. In addition to the well-characterized defects in the Vgamma2 repertoire and functional responses to phosphoantigen, the proportion of CD27-/CD45RA- Vgamma2Vdelta2 T cells after isopentenyl pyrophosphate stimulation was reduced sharply in HIV+ donors versus controls. Thus, HIV infection has multiple impacts on the circulating Vgamma2Vdelta2 T cell population that combine to reduce the potential effector activity in terms of tumor cytotoxicity. Changes in Vgamma2Vdelta2 T cells, along with concomitant effects on NK and NKT cells that also contribute to tumor surveillance, may be important factors for elevating the risk of malignancy during AIDS.

Circulating natural killer and gammadelta T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus.

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and gammadelta T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and gammadelta T cells, indicating that this is not a pathogenic event. V(3)9 T cells, representing the majority of circulating gammadelta T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Altered cord blood gammadelta T cell repertoire in Nigeria: possible impacts of environmental factors on neonatal immunity.

Infectious diseases during pregnancy can impact the development of fetal immunity, leading to reduced neonatal resistance to infection and decreased responses to pediatric vaccines. Plasmodium falciparum causes placental infection in low parity pregnant women and is among the pathogens that affect fetal immunity. Recognizing the relationship between malaria and gammadelta T lymphocytes in adults, we asked whether neonatal gammadelta T cells would be altered in malaria-endemic regions as a marker for changes in fetal immunity. Our initial studies compared cord blood gammadelta T cells from deliveries to HIV- mothers in Jos (Nigeria) where malaria is endemic, or in Rome (Italy). We noted substantial differences in the Vgamma2 repertoire for cord blood collected in Jos or Rome; differences were consistent with a negative selection mechanism operating on the fetal Vgamma2 chain repertoire in neonates from Jos. A specific disruption affected the fraction of gammadelta T cells that we expect will respond to Bacille Calmette-Guerin (BCG). Fetal gammadelta T cell depletion might be a mechanism for impaired neonatal immunity and lowered responses to pediatric vaccines.